Although the interval from the midcycle gonadotropin surge to follicle rupture in primates is 36-42 hours, events culminating in ovulation are poorly understood. As part of our efforts to elucidate this sequence of events, steroidogenic activity and expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were examined in follicles before and after administering an ovulatory bolus of recombinant human chorionic gonadotropin (hCG). Adult rhesus monkeys were treated with recombinant gonadotropins (Ares Serono) to promote multiple follicular development. Large (r4mm) and small (s3mm) antral follicles were aspirated before (0 hr) and after (24 hr) injection of rhCG (Ares Serono). Granulosa cells (GCs) were cultured in the absence (controls) or presence of pregnenolone, low density lipoprotein (LDL), hCG, or the soluble cholesterol analog, 25-hydroxycholesterol (OHC). RNA was isolated from GCs for reverse transcription-polymerase chain reaction (RT-PCR) assay of MMPs and TIMPs. Follicular fluid (FF) was analyzed for MMP and inhibitor activity by zymography and protease inhibitor assays, respectively. GCs from large follicles produced 20-fold (p<0.001) greater levels of progesterone (P) during control culture when isolated 24 hr after versus before the hCG bolus. GCs also exhibited an increased (p<0.01) response to hCG, LDL, and OHC in terms of P production in vitro when obtained after the in vivo hCG bolus. The mRNAs for gelatinase A, TIMP-1, and TIMP-2 were detected by RT-PCR in GCs at both time points. Zymographic analysis demonstrated the presence of a 72-kDa protease (gelatinase A) in FF, but no apparent differences were observed before and after hCG in vivo. Likewise, there was no change in protease inhibitory activity in FF between 0 and 24 hr hCG exposure. Therefore, different regulatory pathways may control changes in steroidogenic and proteolytic activity in the periovulatory follicle following the gonadotropin surge in primates.